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1.
Mol Cancer Ther ; 2019 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-31712395

RESUMO

Genetic alterations in tumor cells provide promising targets for anti-tumor therapy. Recently, loss of methylthioadenosine phosphorylase (MTAP), a deletion frequently occurring in cancer, has been shown to create vulnerability to the inhibition of the protein arginine methyltransferase 5 (PRMT5). MTAP deficiency leads to accumulation of methylthioadenosine (MTA), which reduces PRMT5 activity, and thus, sensitizes the tumor cells to selective PRMT5 inhibitors (PRMT5i). PRMT5i are investigated as a new strategy to selectively kill MTAP-deficient tumor cells by blocking residual PRMT5 activity, but also to treat PRMT5-overexpressing tumors. Though many studies investigated the role of PRMT5 in cancer, only little data exist about the effect of PRMT5 inhibition on immune cells. As we could show that the tumor metabolite MTA suppresses T-cells, we asked if selective PRMT5 inhibition is detrimental for T-cell immune responses. Therefore, we examined the effect of the synthetic PRMT5 inhibitor EPZ015666 on human CD8+ T-cells in direct comparison to the naturally occurring PRMT5-inhibiting molecule MTA. Both compounds reduced T-cell proliferation, viability and functionality. In addition, T cell metabolism was impaired upon PRMT5 inhibition. These effects coincided with the induction of p53 expression and reduced AKT/mTOR signaling. Our data clearly demonstrate that PRMT5 activity is involved in various cellular processes of human CD8+ T-cells associated with essential T-cell functions. Therefore, not only tumor cells but also anti-tumor immune responses are compromised by PRMT5 inhibitors. This emphasizes the importance of considering side effects on the immune system when developing new strategies to specifically target not only MTAP-deficient tumors.

2.
Front Immunol ; 10: 883, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31105702

RESUMO

The recently discovered population of TCRαß+ CD4-/CD8- (double-negative, DN) T-cells are highly potent suppressor cells in mice and humans. In preclinical transplantation models, adoptive transfer of DN T-cells specifically inhibits alloreactive T-cells and prevents transplant rejection or graft-vs.-host disease (GvHD). Interestingly, clinical studies in patients who underwent allogeneic stem cell transplantation reveal an inverse correlation between the frequency of circulating DN T-cells and the severity of GvHD, suggesting a therapeutic potential of human DN T-cells. However, their exact mode of action has not been elucidated yet. Investigating the impact of DN T-cells on conventional T-cells, we found that human DN T-cells selectively inhibit mTOR signaling in CD4 T-cells. Given that mTOR is a critical regulator of cellular metabolism, we further determined the impact of DN T-cells on the metabolic framework of T-cells. Intriguingly, DN T-cells diminished expression of glucose transporters and glucose uptake, whereas fatty acid uptake was not modified, indicating that DN T-cells prevent metabolic adaptation of CD4 T-cells upon activation (i.e., glycolytic switch) thereby contributing to their suppression. Further analyses demonstrated that CD4 T-cells also do not upregulate homing receptors associated with inflammatory processes. In contrast, expression of central memory-cell associated cell surface markers and transcription factors were increased by DN T-cells. Moreover, CD4 T-cells failed to produce inflammatory cytokines after co-culture with DN T-cells, whereas IL-2 secretion was enhanced. Taken together DN T-cells impair metabolic reprogramming of conventional CD4 T-cells by abrogating mTOR signaling, thereby modulating CD4 T-cell functionality. These results uncover a new mechanism of DN T-cell-mediated suppression, pointing out that DN T-cells could serve as cell-based therapy to limit alloreactive immune response.

3.
J Cell Sci ; 132(6)2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30745335

RESUMO

The four and a half LIM domains protein 2 (Fhl2) is an adaptor protein capable of mediating protein-protein interactions. Here, we report for the first time phenotypic changes in the brain of Fhl2-deficient mice. We showed that Fhl2 is expressed in neural stem cells, precursors and mature cells of neuronal lineage. Moreover, Fhl2 deficiency leads to delayed neuroblast migration in vivo, premature astroglial differentiation of neural stem cells (NSCs) in vitro, and a gliosis-like accumulation of glial fibrillary acidic protein (GFAP)-positive astrocytes in vivo that substantially increases with age. Collectively, Fhl2-deficiency in the brain interrupts the maintenance and the balanced differentiation of adult NSCs, resulting in preferentially glial differentiation and early exhaustion of the NSC pool required for adult neurogenesis.

4.
J Immunother Cancer ; 6(1): 116, 2018 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-30396365

RESUMO

Acute myeloid leukemia (AML) is the most common acute leukemia amongst adults with a 5-year overall survival lower than 30%. Emerging evidence suggest that immune alterations favor leukemogenesis and/or AML relapse thereby negatively impacting disease outcome. Over the last years myeloid derived suppressor cells (MDSCs) have been gaining momentum in the field of cancer research. MDSCs are a heterogeneous cell population morphologically resembling either monocytes or granulocytes and sharing some key features including myeloid origin, aberrant (immature) phenotype, and immunosuppressive activity. Increasing evidence suggests that accumulating MDSCs are involved in hampering anti-tumor immune responses and immune-based therapies. Here, we demonstrate increased frequencies of CD14+ monocytic MDSCs in newly diagnosed AML that co-express CD33 but lack HLA-DR (HLA-DRlo). AML-blasts induce HLA-DRlo cells from healthy donor-derived monocytes in vitro that suppress T-cells and express indoleamine-2,3-dioxygenase (IDO). We investigated whether a CD33/CD3-bispecific BiTE® antibody construct (AMG 330) with pre-clinical activity against AML-blasts by redirection of T-cells can eradicate CD33+ MDSCs. In fact, T-cells eliminate IDO+CD33+ MDSCs in the presence of AMG 330. Depletion of total CD14+ cells (including MDSCs) in peripheral blood mononuclear cells from AML patients did not enhance AMG 330-triggered T-cell activation and expansion, but boosted AML-blast lysis. This finding was corroborated in experiments showing that adding MDSCs into co-cultures of T- and AML-cells reduced AML-blast killing, while IDO inhibition promotes AMG 330-mediated clearance of AML-blasts. Taken together, our results suggest that AMG 330 may achieve anti-leukemic efficacy not only through T-cell-mediated cytotoxicity against AML-blasts but also against CD33+ MDSCs, suggesting that it is worth exploring the predictive role of MDSCs for responsiveness towards an AMG 330-based therapy.


Assuntos
Leucemia Mieloide Aguda/imunologia , Monócitos/metabolismo , Células Supressoras Mieloides/imunologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Feminino , Humanos , Leucemia Mieloide Aguda/patologia , Masculino
5.
Transfusion ; 58(9): 2175-2183, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30204947

RESUMO

BACKGROUND: With the discontinuation of the last generation of apheresis machines, new options for monocyte apheresis became available. As apheresis products play a crucial role in the generation of new cellular therapeutics (e.g., generation of dendritic cells [DCs] or precursor for T-cell experiments) we sought to compare two different collection programs for potential benefits or disadvantages due to different composition of the cellular products. STUDY DESIGN AND METHODS: Composition of discontinuously and continuously collected monocyte products from the same 13 donors was analyzed. For further evaluation as starting material for manufacturing of cellular therapeutics typically used steps such as Ficoll Isopaque, cryoconservation and monocyte isolation, with subsequent generation of mature DCs (mDCs) and assessment of T-cell function, were performed on seven of these apheresis pairs. RESULTS: Yield of total cells, monocytes, and mDCs was equal with both methods. T-cell composition did not differ significantly in content of CD3+, CD4+, and CD8+ cells. Differentiation status and cytokine production of CD8+ T cells upon stimulation with cytomegalovirus pp65 antigen was not significantly different. CONCLUSION: Both methods seem comparably suited for the generation of cellular products. If the intended use is "fresh" (without cryoconservation), continuously harvested cells show better cell numbers, while discontinuously harvested cells show better recovery after cryoconservation.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Leucaférese/métodos , Monócitos , Doadores de Sangue , Preservação de Sangue , Células Cultivadas , Criopreservação , Células Dendríticas/citologia , Citometria de Fluxo , Humanos , Linfocinas/metabolismo , Monócitos/citologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo
6.
Sci Rep ; 8(1): 9401, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29925980

RESUMO

Biomimetic scaffolds are of great interest to tissue engineering (TE) and tissue repair as they support important cell functions. Scaffold coating with soluble collagen-I has been used to achieve better tissue integration in orthopaedy, however, as collagen persistence was only temporary such efforts were limited. Adequate coverage with cell-derived ECM collagen-I would promise great success, in particular for TE of mechanically challenged tissues. Here, we have used label-free, non-invasive multiphoton microscopy (MPM) to characterise bacterial nanocellulose (BNC) - a promising biomaterial for bone TE - and their potency to stimulate collagen-I formation by mesenchymal stem cells (MSCs). BNC fleeces were investigated by Second Harmonic Generation (SHG) imaging and by their characteristic autofluorescence (AF) pattern, here described for the first time. Seeded MSCs adhered fast, tight and very stable, grew to multilayers and formed characteristic, wide-spread and long-lasting collagen-I. MSCs used micron-sized lacunae and cracks on the BNC surface as cell niches. Detailed analysis using a collagen-I specific binding protein revealed a highly ordered collagen network structure at the cell-material interface. In addition, we have evidence that BNC is able to stimulate MSCs towards osteogenic differentiation. These findings offer new options for the development of engineered tissue constructs based on BNC.


Assuntos
Materiais Biocompatíveis/farmacologia , Celulose/farmacologia , Células-Tronco Mesenquimais/citologia , Engenharia Tecidual/métodos , Fosfatase Alcalina/metabolismo , Materiais Biocompatíveis/química , Celulose/química , Colágeno Tipo I/metabolismo , Citometria de Fluxo , Células-Tronco Mesenquimais/efeitos dos fármacos , Microscopia
7.
J Clin Invest ; 128(3): 916-930, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29376889

RESUMO

Acute graft-versus-host disease (GVHD) represents a severe, T cell-driven inflammatory complication following allogeneic hematopoietic cell transplantation (allo-HCT). GVHD often affects the intestine and is associated with a poor prognosis. Although frequently detectable, proinflammatory mechanisms exerted by intestinal tissue-infiltrating Th cell subsets remain to be fully elucidated. Here, we show that the Th17-defining transcription factor basic leucine zipper transcription factor ATF-like (BATF) was strongly regulated across human and mouse intestinal GVHD tissues. Studies in complete MHC-mismatched and minor histocompatibility-mismatched (miHA-mismatched) GVHD models revealed that BATF-expressing T cells were functionally indispensable for intestinal GVHD manifestation. Mechanistically, BATF controlled the formation of colon-infiltrating, IL-7 receptor-positive (IL-7R+), granulocyte-macrophage colony-stimulating factor-positive (GM-CSF+), donor T effector memory (Tem) cells. This T cell subset was sufficient to promote intestinal GVHD, while its occurrence was largely dependent on T cell-intrinsic BATF expression, required IL-7-IL-7R interaction, and was enhanced by GM-CSF. Thus, this study identifies BATF-dependent pathogenic GM-CSF+ effector T cells as critical promoters of intestinal inflammation in GVHD and hence putatively provides mechanistic insight into inflammatory processes previously assumed to be selectively Th17 driven.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Doença Enxerto-Hospedeiro/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Intestinos/patologia , Subpopulações de Linfócitos T/metabolismo , Animais , Biópsia , Colo/patologia , Feminino , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Inflamação , Intestinos/imunologia , Complexo Principal de Histocompatibilidade , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Prognóstico , Transplante Homólogo
8.
Nano Lett ; 18(1): 513-519, 2018 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-29227108

RESUMO

Cellular secretion of proteins into the extracellular environment is an essential mediator of critical biological mechanisms, including cell-to-cell communication, immunological response, targeted delivery, and differentiation. Here, we report a novel methodology that allows for the real-time detection and imaging of single unlabeled proteins that are secreted from individual living cells. This is accomplished via interferometric detection of scattered light (iSCAT) and is demonstrated with Laz388 cells, an Epstein-Barr virus (EBV)-transformed B cell line. We find that single Laz388 cells actively secrete IgG antibodies at a rate of the order of 100 molecules per second. Intriguingly, we also find that other proteins and particles spanning ca. 100 kDa-1 MDa are secreted from the Laz388 cells in tandem with IgG antibody release, likely arising from EBV-related viral proteins. The technique is general and, as we show, can also be applied to studying the lysate of a single cell. Our results establish label-free iSCAT imaging as a powerful tool for studying the real-time exchange between cells and their immediate environment with single-protein sensitivity.


Assuntos
Linfócitos B/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Imunoglobulina G/análise , Análise de Célula Única/métodos , Linfócitos B/virologia , Linhagem Celular , Herpesvirus Humano 4/isolamento & purificação , Humanos , Imunoglobulina G/imunologia , Luz , Microscopia de Interferência/métodos , Imagem Óptica/métodos , Espalhamento de Radiação
9.
Oncoimmunology ; 5(8): e1184802, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27622058

RESUMO

The immunosuppressive tumor microenvironment represents one of the main obstacles for immunotherapy of cancer. The tumor milieu is among others shaped by tumor metabolites such as 5'-deoxy-5'-methylthioadenosine (MTA). Increased intratumoral MTA levels result from a lack of the MTA-catabolizing enzyme methylthioadenosine phosphorylase (MTAP) in tumor cells and are found in various tumor entities. Here, we demonstrate that MTA suppresses proliferation, activation, differentiation, and effector function of antigen-specific T cells without eliciting cell death. Conversely, if MTA is added to highly activated T cells, MTA exerts cytotoxic effects on T cells. We identified the Akt pathway, a critical signal pathway for T cell activation, as a target of MTA, while, for example, p38 remained unaffected. Next, we provide evidence that MTA exerts its immunosuppressive effects by interfering with protein methylation in T cells. To confirm the relevance of the suppressive effects of exogenously added MTA on human T cells, we used an MTAP-deficient tumor cell-line that was stably transfected with the MTAP-coding sequence. We observed that T cells stimulated with MTAP-transfected tumor cells revealed a higher proliferative capacity compared to T cells stimulated with Mock-transfected cells. In conclusion, our findings reveal a novel immune evasion strategy of human tumor cells that could be of interest for therapeutic targeting.

10.
Blood ; 128(2): 227-38, 2016 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-27099149

RESUMO

Autoimmune lymphoproliferative syndrome (ALPS) is a human disorder characterized by defective Fas signaling, resulting in chronic benign lymphoproliferation and accumulation of TCRαß(+) CD4(-) CD8(-) double-negative T (DNT) cells. Although their phenotype resembles that of terminally differentiated or exhausted T cells, lack of KLRG1, high eomesodermin, and marginal T-bet expression point instead to a long-lived memory state with potent proliferative capacity. Here we show that despite their terminally differentiated phenotype, human ALPS DNT cells exhibit substantial mitotic activity in vivo. Notably, hyperproliferation of ALPS DNT cells is associated with increased basal and activation-induced phosphorylation of serine-threonine kinases Akt and mechanistic target of rapamycin (mTOR). The mTOR inhibitor rapamycin abrogated survival and proliferation of ALPS DNT cells, but not of CD4(+) or CD8(+) T cells in vitro. In vivo, mTOR inhibition reduced proliferation and abnormal differentiation by DNT cells. Importantly, increased mitotic activity and hyperactive mTOR signaling was also observed in recently defined CD4(+) or CD8(+) precursor DNT cells, and mTOR inhibition specifically reduced these cells in vivo, indicating abnormal programming of Fas-deficient T cells before the DNT stage. Thus, our results identify the mTOR pathway as a major regulator of lymphoproliferation and aberrant differentiation in ALPS.


Assuntos
Síndrome Linfoproliferativa Autoimune/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Transdução de Sinais/imunologia , Serina-Treonina Quinases TOR/imunologia , Adolescente , Adulto , Síndrome Linfoproliferativa Autoimune/patologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Criança , Pré-Escolar , Feminino , Humanos , Lectinas Tipo C/imunologia , Antígenos Comuns de Leucócito/imunologia , Masculino , Proteínas Proto-Oncogênicas c-akt/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transativadores/imunologia
11.
J Immunol ; 195(7): 3139-48, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26324773

RESUMO

Recently, a novel subset of TCRαß(+) CD4(-) CD8(-) double-negative (DN) T cells was described to suppress immune responses in both mice and humans. Moreover, in murine models, infusion and/or activation of DN T cells specifically suppressed alloreactive T cells and prevented the development of graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. We demonstrated that human DN T cells, like their murine counterparts, are highly potent suppressor cells of both CD4(+) and CD8(+) T cell responses. After hematopoietic stem cell transplantation and other lymphopenic conditions, IL-7 plays an important role in the reconstitution, survival, and homeostasis of the T cell compartment. Because IL-7 was shown to interfere with T cell functionality, we asked whether IL-7 affects the functionality of human DN T cells. Intriguingly, IL-7 diminished the suppressive activity of DN T cells toward allogeneic CD4(+) effector T cells. Of interest, our studies revealed that IL-7 activates the Akt/mechanistic target of rapamycin (mTOR) pathway in human DN T cells. Importantly, selective inhibition of the protein kinases Akt or mTOR reversed the IL-7 effect, thereby restoring the functionality of DN T cells, whereas inhibition of other central T cell signaling pathways did not. Further analyses suggest that the IL-7/Akt/mTOR signaling cascade downregulates anergy-associated genes and upregulates activation- and proliferation-associated factors that may be crucial for DN T cell functionality. These findings indicate that IL-7 and Akt/mTOR signaling are critical factors for the suppressive capacity of DN T cells. Targeting of these pathways by pharmacological agents may restore and/or enhance DN T cell functionality in graft-versus-host disease.


Assuntos
Tolerância Imunológica/imunologia , Interleucina-7/imunologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Serina-Treonina Quinases TOR/imunologia , Antígenos CD4/genética , Linfócitos T CD4-Positivos/imunologia , Antígenos CD8/genética , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Células Cultivadas , Doença Enxerto-Hospedeiro/imunologia , Transplante de Células-Tronco Hematopoéticas , Humanos , Ativação Linfocitária/imunologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Transdução de Sinais/imunologia , Serina-Treonina Quinases TOR/antagonistas & inibidores
12.
Blood ; 124(6): 851-60, 2014 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-24894771

RESUMO

Accumulation of CD3(+) T-cell receptor (TCR)αß(+)CD4(-)CD8(-) double-negative T cells (DNT) is a hallmark of autoimmune lymphoproliferative syndrome (ALPS). DNT origin and differentiation pathways remain controversial. Here we show that human ALPS DNT have features of terminally differentiated effector memory T cells reexpressing CD45RA(+) (TEMRA), but are CD27(+)CD28(+)KLRG1(-) and do not express the transcription factor T-bet. This unique phenotype was also detected among CD4(+) or CD8(+) ALPS TEMRA cells. T-cell receptor ß deep sequencing revealed a significant fraction of shared CDR3 sequences between ALPS DNT and both CD4(+) and CD8(+)TEMRA cells. Moreover, in ALPS patients with a germ line FAS mutation and somatic loss of heterozygosity, in whom biallelic mutant cells can be tracked by absent Fas expression, Fas-negative T cells accumulated not only among DNT, but also among CD4(+) and CD8(+)TEMRA cells. These data indicate that in human Fas deficiency DNT cannot only derive from CD8(+), but also from CD4(+) T cells. Furthermore, defective Fas signaling leads to aberrant transcriptional programs and differentiation of subsets of CD4(+) and CD8(+) T cells. Accumulation of these cells before their double-negative state appears to be an important early event in the pathogenesis of lymphoproliferation in ALPS patients.


Assuntos
Síndrome Linfoproliferativa Autoimune/imunologia , Síndrome Linfoproliferativa Autoimune/patologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Receptor fas/deficiência , Receptor fas/genética , Adolescente , Adulto , Síndrome Linfoproliferativa Autoimune/genética , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Criança , Pré-Escolar , Estudos de Associação Genética , Mutação em Linhagem Germinativa , Humanos , Memória Imunológica , Antígenos Comuns de Leucócito/metabolismo , Perda de Heterozigosidade , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Proteínas com Domínio T/metabolismo , Subpopulações de Linfócitos T/metabolismo , Adulto Jovem
14.
Stem Cells ; 31(8): 1715-25, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23554294

RESUMO

Adoptive transfer of third-party mesenchymal stromal cells (MSCs) has emerged as a promising tool for the treatment of steroid-refractory graft-versus-host disease (GVHD). Despite numerous in vitro studies and preclinical models, little is known about their effects on the patients' immune system. We assessed immune alterations in the T-cell, B-cell, natural killer cell, dendritic cell, and monocytic compartments of steroid-refractory GVHD patients 30, 90, and 180 days after MSC (n = 6) or placebo (n = 5) infusion, respectively. Infused MSCs were bioactive as suggested by the significant reduction in epithelial cell death, which represents a biomarker for acute GVHD. There were several indications that MSCs shift the patients' immune system toward a more tolerogenic profile. Most importantly, infusion of MSCs was associated with increased levels of regulatory (forkhead box P3 (FOXP3)(+) and interleukin (IL)-10(+) ) T-cells, reduced pro-inflammatory IL-17(+) T(Th17)-cells, and skewing toward type-2 T-helper cell responses. Furthermore, IL-2, which has been recently shown to exert a positive immune modulating effect in GVHD patients, was higher in the MSC patients at all evaluated time points during 6 months after MSC-infusion. Overall, our findings will contribute to the refinement of monitoring tools, for assessing MSC treatment-efficacy and increase our understanding regarding the MSCs' in vivo effects.


Assuntos
Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Adulto , Idoso , Feminino , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/patologia , Neoplasias Hematológicas/cirurgia , Humanos , Imunidade Celular/imunologia , Imunoterapia Adotiva/métodos , Masculino , Pessoa de Meia-Idade , Transplante Homólogo
16.
J Immunol Methods ; 344(2): 98-108, 2009 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-19332073

RESUMO

Novel immunosuppressive strategies are targeting for an antigen-specific deletion of T cells responsible for organ damage in autoimmunity and allograft rejection. Here, we present a new flow cytometry-based assay that allows the reliable and efficient detection of T cells that were eliminated in an antigen-specific fashion. A stable cell-labelling technique utilizing the two membrane dyes PKH26 and PKH67 has been combined with annexin V and 7-aminoactinomycin (7-AAD) staining to detect apoptotic cells. A differential gating strategy enabled us to determine the viability/apoptosis for each PKH-stained T cell subpopulation independently. The capability to simultaneously analyze apoptosis within T cell mixtures of different antigen specificities establishes this assay as a superior tool for the further development of novel antigen-specific immunosuppressive approaches.


Assuntos
Apoptose , Membrana Celular/química , Citometria de Fluxo/métodos , Subpopulações de Linfócitos T/metabolismo , Anexina A5/química , Células Apresentadoras de Antígenos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Dactinomicina/análogos & derivados , Dactinomicina/química , Corantes Fluorescentes/química , Humanos , Células Jurkat , Compostos Orgânicos/química , Sensibilidade e Especificidade
17.
J Biol Chem ; 280(26): 24356-62, 2005 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15857826

RESUMO

Tumor necrosis factor-alpha (TNFalpha) is a potent inhibitor of renin gene expression in renal juxtaglomerular cells. We have found that TNFalpha suppresses renin transcription via transcription factor NFkappaB, which targets a cAMP responsive element (CRE) in the renin promoter. Here we aimed to further clarify the role of NFkappaB and the canonical CRE-binding proteins of the CRE-binding protein/activating transcription factor (CREB/ATF) family in the inhibition of renin gene expression by TNFalpha in the juxtaglomerular cell line As4.1. TNFalpha caused a moderate decrease in the binding of CREB1 to its cognate CRE DNA binding site. On the other hand, NFkappaB-p65 transcriptional activity was substantially reduced by TNFalpha, which targeted a trans-activation domain at the very C terminus of the p65 molecule. Our results suggest that TNFalpha inhibits renin gene expression by decreasing the transactivating capacity of NFkappaB-p65 and partially by attenuating CREB1 binding to CRE.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Regulação para Baixo , NF-kappa B/fisiologia , Renina/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Animais , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , DNA/metabolismo , Ensaio de Imunoadsorção Enzimática , Rim/metabolismo , Luciferases/metabolismo , Camundongos , Mutação , NF-kappa B/metabolismo , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , RNA/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , Renina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição RelA , Fatores de Transcrição/metabolismo , Transcrição Genética , Ativação Transcricional , Transfecção
18.
J Biol Chem ; 279(2): 1458-67, 2004 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-14563845

RESUMO

Tumor necrosis factor-alpha (TNFalpha) is known to inhibit renin gene expression in juxtaglomerular cells, which are the main source of renin in vivo. In the present study we aimed to characterize the intracellular mechanisms of TNFalpha signaling to renin gene in the mouse juxtaglomerular cell line As4.1. TNFalpha was found to activate NFkappaB, which is one of the principal intracellular mediators of TNFalpha signal transduction. Constitutive activation of NFkappaB suppressed renin gene transcription, but NFkappaB appeared not to target the NFkappaB binding sites in the renin promoter. Thus, NFkappaB, but not the canonical NFkappaB binding sequences in the renin promoter, seemed to be involved in the suppression of renin transcription by TNFalpha. Deletion/mutation analysis revealed that the effect of TNFalpha on renin gene is transmitted by a cAMP-responsive element (CRE) located at -2697 to -2690. Mobility shift/supershift assays evidenced for the presence of NFkappaB proteins in the complex that binds to mouse renin CRE. Our results strongly suggest that NFkappaB mediates the effect of TNFalpha on renin transcription targeting a CRE in the mouse renin promoter.


Assuntos
NF-kappa B/metabolismo , Renina/metabolismo , Transcrição Genética , Fator de Necrose Tumoral alfa/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Elementos Facilitadores Genéticos , Immunoblotting , Luciferases/metabolismo , Camundongos , Microscopia de Fluorescência , Modelos Genéticos , Mutação , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , Renina/biossíntese , Renina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Tempo , Ativação Transcricional , Transfecção
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